JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.13.6115–6120.2001

July 2001, p. 6115–6120 Vol. 75, No. 13

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Axonal Damage Is T Cell Mediated and Occurs Concomitantlywith Demyelination in Mice Infected with a

Neurotropic CoronavirusAJAI A. DANDEKAR,1 GREGORY F. WU,2 LECIA PEWE,3 AND STANLEY PERLMAN1,2,3,4*

Interdisciplinary Programs in Immunology1 and Neuroscience2 and Departments of Pediatrics3 and Microbiology,4

University of Iowa, Iowa City, Iowa 52242

Received 10 January 2001/Accepted 28 March 2001

Mice infected with mouse hepatitis virus (MHV) strain JHM develop primary demyelination. Herein weshow that axonal damage occurred in areas of demyelination and also in adjacent areas devoid of myelindamage. Immunodeficient MHV-infected RAG12/2 mice (mice defective in recombinase activating gene 1expression) do not develop demyelination unless they receive splenocytes from a mouse previously immunizedagainst MHV (G. F. Wu, A. Dandekar, L. Pewe, and S. Perlman, J. Immunol. 165:2278–2286, 2000). In thepresent study, we show that adoptive transfer of T cells was also required for the majority of the axonal injuryobserved in these animals. Both demyelination and axonal damage were apparent by 7 days posttransfer.Recent data suggest that axonal injury is a major factor in the long-term disability observed in patients withmultiple sclerosis. Our data demonstrate that immune system-mediated damage to axons is also a commonfeature in mice with MHV-induced demyelination. Remarkably, there appeared to be a minimal, if any, intervalof time between the appearance of demyelination and that of axonal injury.

The hallmark of multiple sclerosis (MS) is the existence ofmultifocal demyelinating plaques within the central nervoussystem (CNS) (15, 23). Although oligodendrocytes, the myelin-producing cells of the CNS, and/or myelin sheaths appear to betargets of immune system-mediated destruction in MS, recentevidence demonstrating that axonal damage also occurs hasrenewed interest in the neuronal correlates of CNS demyeli-nation. Axonal damage in the CNSs of patients with MS wasdemonstrated using immunohistochemical staining for amyloidprecursor protein (5) or nonphosphorylated neurofilament H(NF) (25) and is likely to be a major component of the long-term disability observed in this disease. Axonal damage wasobserved most abundantly in areas of active demyelination buthas also been detected in the white matter adjacent to areas ofdemyelination and in normal-appearing white matter (3, 5, 11,25). In other studies, a decrease in the amount of the neuron-specific compound N-acetylaspartate in areas of demyelinationwas demonstrated by using magnetic resonance spectroscopyimaging (1). N-acetylaspartate was also decreased in normal-appearing white matter, possibly because axons were damagedas they crossed a site of demyelination or inflammation (4).

Axonal damage has been documented in rats with chronicactive experimental autoimmune encephalomyelitis induced byimmunization with myelin-oligodendrocyte glycoprotein (11)and in guinea pigs with chronic experimental autoimmuneencephalomyelitis (17). Axonal loss, confined primarily to me-dium and large myelinated fibers, has also been demonstratedin mice with chronic demyelination induced by Theiler’s mu-rine encephalomyelitis virus (14, 19). Axonal loss correlated

with neurological dysfunction and occurred relatively late inthe disease course, suggesting that only demyelinated axonswere damaged during disease progression.

Rodents infected with mouse hepatitis virus (MHV) strainJHM (MHV-JHM) develop acute and chronic demyelinatingdiseases (10, 12, 21). Although in this model demyelination isprimary with axons mostly spared, infrequent axonal injury atsites of demyelination has also been noted (2, 22). The mech-anism of axonal damage was not examined in any of these priorstudies. Inoculation of immunocompetent C57Bl/6 (B6) micewith a variant of MHV-JHM with diminished tropism for neu-rons, strain 2.2-V-1, resulted in the appearance of demyelina-tion and hind limb paresis at approximately 12 to 15 dayspostinoculation (p.i.) (6, 7). However, 6-week-old severe com-bined immunodeficiency or RAG12/2 mice did not developdemyelination after infection with 2.2-V-1 (9, 28). Demyelina-tion and clinical disease developed in these mice 7 to 8 daysafter adoptive transfer of splenocytes from B6 mice previouslyimmunized with MHV.

The studies described above suggest that axonal damageoccurs early in the process of autoimmune or virus-induceddemyelination, is immune mediated, and is a key factor in theneurological disease that develops in the human or animalhost. Although extensive infiltration of lymphocytes and mac-rophages into the spinal cord is a consistent feature of thedemyelinating lesions in all of these models, little is knownabout the relative importance of T lymphocytes and other cellsin this process. Furthermore, the precise relationship betweenthe onset of demyelination and appearance of axonal damagehas not been well established. In this study, the adoptive trans-fer model of MHV-induced demyelination was used to addressthese issues, since the rapid onset of disease observed in thismodel makes it uniquely suited to analyze the early stages ofaxonal damage.

* Corresponding author. Mailing address: Department of Pediatrics,2042 Medical Laboratories, University of Iowa, Iowa City, IA 52242.Phone: (319) 335-8549. Fax: (319) 335-8991. E-mail: Stanley-Perlman@uiowa.edu.

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MATERIALS AND METHODS

Virus. The neuroattenuated variant of MHV-JHM, 2.2-V-1 (7), was gener-ously provided by J. Fleming (University of Wisconsin, Madison).

Animals. Pathogen-free B6 mice were obtained from the National CancerInstitute (Bethesda, Md.). RAG12/2 mice were obtained from Jackson Labo-ratory (Bar Harbor, Maine). No mature B or T cells were detected in these miceby fluorescence-activated cell sorter analysis using antibodies specific forCD45R/B220, CD4, and CD8 antigens.

Animal model. B6 or RAG12/2 mice were infected with 103 PFU of 2.2-V-1by intracranial injection (26). Adoptive transfer of splenocytes from B6 miceimmunized intraperitoneally with wild-type MHV-JHM to infected RAG12/2mice was performed as previously described (28). Wild-type MHV-JHM wasused for immunization to maximize the anti-MHV immune response in donoranimals. A total of 34 2.2-V-1-infected RAG12/2 mice were used in theseexperiments. Of these 34 mice, 10 did not receive any transferred cells, 16received splenocytes treated with complement alone, and 8 received splenocytesdepleted of both CD4 and CD8 T cells. No infectious virus could be detected byplaque assay in the transferred cells (28).

Antibodies. Monoclonal antibodies (MAbs) SMI-32 and SMI-99 (SternbergerMonoclonal Antibodies, Baltimore, Md.) were used to label nonphosphorylatedNF and myelin basic protein (MBP), respectively. Rat anti-macrophage MAb(F4/80; Serotec, Oxford, England) and MAb to MHV-JHM nucleocapsid (N)protein (5B188.2; kindly provided by M. Buchmeier, The Scripps ResearchInstitute) were used for immunohistochemical labeling of macrophages or mi-croglia and virus antigen, respectively.

Histology. After perfusion with phosphate-buffered saline, brains and spinalcords were fixed in 10% normal buffered formalin in Histochoice fixative (Am-resco, Solon, Ohio) or in zinc formaldehyde (Labsco, Louisville, Ky.). For ex-amination of myelin and cell morphology, 8-mm-thick sections were stained withluxol fast blue.

Immunofluorescence assay. The distribution of nonphosphorylated NF ormyelin was determined as follows. Sections (8 mm thick) from samples fixed withHistochoice or zinc formaldehyde were permeabilized with 0.1% Triton X-100,blocked with CAS BLOCK (Zymed Laboratories, San Francisco, Calif.), andincubated with primary antibody (SMI-32 diluted 1:5,000 to 1:10,000 or SMI-99diluted 1:1,000 in 1% normal goat serum) overnight at 4°C. After the sectionswere washed, they were incubated with fluorescein isothiocyanate-conjugatedgoat anti-mouse immunoglobulin G antibody diluted 1:100 (ICN/Cappel, Au-rora, Ohio) for 1 h at room temperature. No staining was present in the absenceof primary antibody (data not shown).

Immunohistochemistry. Sections were incubated with F4/80 (diluted 1:200) or5B188.2 (diluted 1:2,000) overnight at 4°C. After the sections were washed, theywere incubated with biotinylated goat anti-rat antibody (F4/80) (1:200) or bio-tinylated goat anti-mouse antibody (5B188.2) (Jackson Immunoresearch Labs,West Grove, Pa.) (1:100) for 1 h at room temperature. Following washing,avidin-horseradish peroxidase (Jackson Immunoresearch Labs) (1:1,000) wasapplied for 1 h. The final substrate utilized for the staining reaction was 3,39-diaminobenzidine (Sigma, St. Louis, Mo.). After development, sections werecounterstained with hematoxylin.

Quantification of axonal damage. (i) Method 1. Spinal cord sections wereexamined using a Zeiss LSM510 confocal microscope. Quantification of axonaldamage was performed as follows. Spinal cords were prepared and stained withantibody to nonphosphorylated NF. Using Vtrace software (Image AnalysisFacility, University of Iowa), images from the white matter of an entire midsag-ittal section were digitized and analyzed. Damaged axons were identified asaccumulations of NF in the white matter, and the number of pixels above thebackground level was recorded. Sections were analyzed in a blind fashion by twoobservers. A background level of staining was identified for each section visuallyusing an area of white matter devoid of any SMI-32 immunoreactivity. Thepercentage of SMI-32-positive pixels per area of white matter in each section wasdetermined by dividing the number of pixels above the threshold level in anaverage of 48 (range, 14 to 107) separate fields (magnification of 320) by thetotal area of white matter.

(ii) Method 2. To compare the amount of axonal damage in demyelinated andnormally myelinated areas, the number of SMI-32-immunoreactive axons permicroscope field at a magnification of 320 was determined using a Leitz DMRBmicroscope with a fluorescence attachment. Twelve fields (four in areas ofdemyelination, four in adjacent areas, and four in normal-appearing white mat-ter) were examined per spinal cord.

Statistical analysis. P values were calculated by using Student’s t test.

RESULTS

Axonal damage was observed following infection with 2.2-V-1. Immunocompetent B6 mice infected with the attenuated2.2-V-1 variant of MHV-JHM develop primary demyelinationwithin 12 days of inoculation, with relative preservation ofaxons (9, 26). In preliminary experiments, we analyzed thesemice with demyelination for axonal damage using antibody tononphosphorylated NF (SMI-32). In uninfected animals, non-phosphorylated NF is present in neuronal cell bodies and den-drites but is not detected in normal white matter (13). SMI-32immunoreactivity was detected in the spinal cords of MHV-infected mice with demyelination, consistent with axonal dam-age (data not shown). In these immunocompetent mice, axonaldamage could result from direct virus damage to axons or glialcells or could be immune mediated. To distinguish these pos-sibilities, axonal damage was assayed in 2.2-V-1-infectedRAG12/2 mice. We showed previously that 2.2-V-1-infectedRAG12/2 mice do not develop demyelination in the absenceof transferred splenocytes. Adoptive transfer of either MHV-specific CD4 or CD8 T cells was sufficient for the developmentof demyelination (27).

Initially, serial sections from naive and MHV-infectedRAG12/2 mice were analyzed for axonal damage, macro-phages or microglia, viral antigen, and myelin integrity. Essen-tially no SMI-32 immunoreactivity was detected in naiveRAG12/2mice (Fig. 1D). However, staining for nonphospho-

FIG. 1. Axonal damage was detected in 2.2-V-1-infectedRAG12/2 mice, but not in uninfected RAG12/2 mice. Spinal cordswere harvested from 2.2-V-1-infected (A to C) and uninfected (D)RAG12/2 mice. Samples were analyzed for demyelination (A), virusantigen (B), and SMI-32 immunoreactivity (C and D). Although viralantigen was abundant in the white and gray matter (B), only occasionalareas of axonal debris were detected in 2.2-V-1-infected RAG12/2mice (C). No SMI-32 immunoreactivity was detected in the whitematter of uninfected mice (D). Ten 2.2-V-1-infected and four unin-fected RAG12/2 mice were used in these experiments. Bar, 100 mm.

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rylated NF was detected at low levels in the spinal cords ofMHV-infected RAG12/2 mice at 10 days p.i. (Fig. 1C), al-though demyelination was not detected in these mice (Fig.1A). Extensive viral replication within both the gray and whitematter was present in the CNSs of these mice (Fig. 1B), show-ing that virus infection in the absence of T and B cells resultedin only a small amount of axonal damage.

Axonal damage was largely immune mediated. Adoptivetransfer of splenocytes from immunized donors to 2.2-V-1-infected RAG12/2 mice resulted in frank demyelination by 7days posttransfer (p.t.) (28). In these experiments, immunesplenocytes were transferred to RAG12/2 mice 3 or 4 daysafter intracerebral inoculation with 2.2-V-1. SMI-32 immuno-reactivity was markedly increased in the spinal cords of thesemice, particularly in areas of demyelination (Fig. 2A and J).Several patterns of SMI-32 immunoreactivity were present inthese mice (Fig. 2J), similar to those observed in the CNSs ofpatients with MS (25). Collections of SMI-32 staining with theappearance of debris, consistent with axonal destruction, weredetected in areas of demyelination. Some axons exhibited dis-continuous staining with focally enlarged caliber, consistentwith degenerative changes. Ovoid bodies attached to axonalremnants, suggesting axonal transection, were also observed(e.g., inset in Fig. 2J). Nonphosphorylated NF is also present inintact demyelinated axons (25), and some of the SMI-32-im-munoreactive axons appeared to be normal axons lacking my-elin. Macrophages or microglia, believed to be the predomi-nant effector cells in MHV-induced demyelination, wereabundant in areas of demyelination and axonal damage (Fig.2D). Viral antigen was also present (Fig. 2G) but at lowerlevels than in mice not receiving transferred splenocytes.

SMI-32 immunoreactivity was also detected in areas adja-cent to demyelinating lesions (Fig. 2K). The myelin in theseareas appeared relatively unaffected after staining with luxolfast blue (Fig. 2B) or with antibody to MBP (data not shown).However, even in these regions of the spinal cord, the whitematter was not truly normal, since activated macrophages weredetected in the general vicinity of the SMI-32-immunoreactiveaxons (Fig. 2E) and viral antigen was detected (Fig. 2H). Thisresult suggested either that the inflammatory process was in itsearly stages in these areas or that axonal damage was a sec-ondary effect of axonal passage through a site of demyelinationor inflammation. No SMI-32 immunoreactivity was detected insome areas of normal-appearing white matter (Fig. 2L), but inthese areas, virus, macrophage, and myelin disruption were notdetected (Fig. 2C, F, and I).

Two approaches were taken to quantify the amount of ax-onal damage present in infected spinal cords. In the first ap-proach, all the white matter was analyzed for SMI-32 immu-noreactivity by confocal microscopy as described in Materialsand Methods (Fig. 3A). This method facilitated comparisonbetween 2.2-V-1-infected RAG12/2 mice that did not receivetransferred cells and those that did. Transfer of immunesplenocytes resulted in a significant increase in axonal damage.Since only about 10 to 15% of the white matter exhibiteddemyelination after adoptive transfer (27) and most axonaldamage was observed in areas of myelin damage, this approachtended to blunt the differences in axonal damage observedamong the various groups. Notably, only low levels of demy-elination (27) or axonal damage (Fig. 3A) were detected when

MHV-infected RAG12/2 mice received splenocytes depletedof both CD4 and CD8 T cells.

In a second approach, the number of damaged axons perunit area was determined in areas of demyelination and inareas of normal-appearing white matter within a single spinalcord by fluorescence microscopy (Fig. 3B). The latter weredivided into areas with early signs of damage (Fig. 2B, E, H,and K) and those that appeared completely normal (Fig. 2C, F,I, and L). This analysis showed that axonal damage occurredpreferentially in areas of demyelination.

Axonal damage was not detected prior to the development ofdemyelination. These results indicated that axonal damage wasdetected by day 7 to 8 p.i., at approximately the same time thatdemyelination was first observed. However, activated macro-phages or microglia were detected at sites of viral infection inthe spinal cord as early as five days p.t., although frank demy-elination was not detected at this time (28). To investigatefurther the relationship between demyelination, macrophageor microglia infiltration and axonal damage, spinal cords wereharvested from mice 4.5 days p.t., and serial sections wereanalyzed as described above. Macrophage infiltration into thewhite matter (Fig. 4B) was detected at sites of viral infection(Fig. 4C). These regions showed little evidence of myelin dam-age when examined by luxol fast blue staining (Fig. 4A) or bystaining with antibody to MBP (data not shown). However, lowlevels of SMI-32 immunoreactivity were detected in these sam-ples (Fig. 4D), suggesting that axonal injury occurred duringearly stages of damage to the myelin sheath. The number ofdamaged axons in areas of macrophage infiltration (12.8 6 1.7damaged axons/mm2) and in areas lacking macrophage infil-tration (2.3 6 0.9 damaged axons/mm2) were significantly dif-ferent (P , 0.001) when the spinal cords of four mice wereanalyzed 4.5 days p.t.

DISCUSSION

These data show that axonal damage was readily detected inthe spinal cords of MHV-infected mice. Axonal damage waspartly T and B cell independent, since low levels were detectedin infected RAG12/2 mice in the absence of adoptive transferof immune splenocytes. However, substantial increases inSMI-32 immunoreactivity were detected following adoptivetransfer, nearly coincident with the onset of demyelination. Anincrease in SMI-32 immunoreactivity was also detected in ar-eas of macrophage infiltration prior to the development ofovert demyelination (Fig. 4). It is likely that these were sites ofincipient myelin damage and axonal injury. The results of thisstudy are consistent with those of other studies suggesting thataxonal damage occurred in the intense inflammatory milieupresent at sites of demyelination but also showed for the firsttime that this damage occurred very early in the disease pro-cess.

We showed previously that demyelination was mediated byT cells in the adoptive transfer model (27). As shown above,axonal damage was not increased if CD4 and CD8 T cells wereremoved prior to adoptive transfer (Fig. 3A). Clinical diseaseand demyelination occurred only occasionally in mice thatreceived splenocytes from donors not previously immunized toMHV, suggesting that MHV-specific T cells were critical fordemyelination to develop (28). Our results suggest that axonal

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FIG. 2. Correlation of axonal damage with demyelination and macrophage infiltration. Spinal cords were harvested from RAG12/2 mice 7days p.t. of immune splenocytes. Serial sections (8 mm thick) were analyzed for demyelination (A to C), macrophages or microglia (D to F), viralantigen (G to I), and nonphosphorylated NF (J to L). Many SMI-32-immunoreactive axons (J) were detected in areas of demyelination (A). Inpanel J, cellular debris (arrowhead), a degenerating axon (large arrow), and an intact demyelinated axon (small arrow) are indicated. Abundantmacrophage infiltration (D) and virus antigen (G) were detected in these lesions. Examination of areas adjacent to the demyelinating lesions(periplaque) revealed relatively normal-appearing white matter (B), infiltration of macrophages (E), presence of viral antigen (H), and reducedamounts of SMI-32 immunoreactivity (K). Finally, more-distal areas of normal-appearing white matter (NAWM) (C) exhibited no macrophageinfiltration (F) or viral antigen (I), with only low levels of SMI-32 immunoreactivity (L). An ovoid body associated with an axonal remnantconsistent with axonal transection is shown in the inset in panel J. Eleven 2.2-V-1-infected RAG12/2 mice analyzed 7 days p.t. were used in theseexperiments. Abbreviations: LFB, luxol fast blue; w, white matter; g, gray matter. Bar, 100 mm.

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damage was also mediated by MHV-specific T cells, at least tothe extent that they were required for initiating the inflamma-tory response that resulted in demyelination.

These results do not eliminate a role for B cells and splenicmacrophages in demyelination or axonal injury but do showthat they are not able to cause demyelination in the absence ofT cells. It is likely that transferred macrophages are not nec-essary for demyelination to develop. In MHV-infected B6mice, chemical depletion of bloodborne macrophages did notresult in a decrease in demyelination (29), suggesting thatmicroglia or perivascular macrophages were able to serve asthe final effector cells of demyelination.

Our results revealed a low level of SMI-32 immunoreactivityin 2.2-V-1-infected RAG12/2 mice in the absence of adop-tively transferred splenocytes. Axonal damage may be a directconsequence of viral infection of neurons or, alternatively,result from infection (and subsequent dysfunction) of glialcells, including oligodendrocytes. In mice deficient in the ex-pression of proteolipid protein, severe axonal pathology is de-tected, although myelin disruption is minimal and overt demy-elination is not detected (8). Damage may also be mediated byNK cells or other parts of the immune system intact inRAG12/2 mice. After adoptive transfer of immune spleno-cytes, axonal damage was most abundant at sites of demyeli-nation, suggesting that the inflammatory response at sites ofdemyelination, and not immune-mediated clearance from neu-rons, was responsible for this increase in damage. In support of

this, axonal damage was detected in B6 mice chronically in-fected with wild-type MHV-JHM (data not shown). Minimalamounts of virus were detected in the gray matter in these mice(16, 24), indicating that the process of neuronal clearance wasaccomplished much earlier. Furthermore, axonal damage wasobserved following infection of B6 mice with 2.2-V-1 (data notshown), even though replication is largely restricted to thewhite matter in these animals (6, 7).

SMI-32 immunoreactivity may represent reversible or irre-versible axonal damage. Axonal transection is irreversible andaccounted for some of the SMI-32 staining that we observed.However, both remyelinated and demyelinated axons may re-cover function via restoration of conduction, possibly second-ary to restoration of sodium channel function (20). Axonaldysfunction resulting from impaired axonal conduction in theCNSs of mice infected with Theiler’s murine encephalomyelitisvirus has been reported and shown to be CD8 T cell mediated(18). In the absence of CD8 T cells, redistribution of ionchannels was detected, thereby preserving neurological func-tion, even in the presence of demyelination.

Our experiments do not address the role of axonal damagein long-term disease progression, since we analyzed mice atearly times after adoptive transfer. However, they showed thataxonal damage was, in large part, immune mediated in miceinfected with MHV and occurs concomitantly with demyelina-tion. This model system will be useful for determining the

FIG. 3. Quantification of increase in SMI-32 reactivity in RAG12/2 mice following adoptive transfer. The amount of axonal damage withinthe white matter of spinal cords from 2.2-V-1-infected RAG12/2 mice was quantified by using two separate methods as described in Materialsand Methods. (A) In the first method, the amount of SMI-32 immunoreactivity within the entire white matter of individual spinal cords wasdetermined. Eight mice were analyzed prior to adoptive transfer (10 days p.i.). Three days p.i., eight mice received undepleted splenocytepopulations, whereas an additional eight mice received splenocytes depleted of both CD4 and CD8 T cells. These mice were harvested 7 days p.t.Three uninfected mice were also analyzed. The amount of SMI-32 immunoreactivity in naive mice was significantly less than that detected in anyof the virus-infected mice (P , 0.01). The amount of staining in mice in the absence of adoptive transfer or in mice that received splenocytesdepleted of both CD4 and CD8 T cells was significantly less than in mice receiving undepleted populations (P , 0.001). There was no statisticaldifference in staining between infected mice receiving doubly depleted cells and those analyzed in the absence of adoptive transfer (P . 0.05). (B)In the second method, axonal damage in areas of demyelination (Demyel.), periplaque, and more-distal normal-appearing white matter (NAWM),as described in the legend to Fig. 2, was quantified by fluorescence microscopy. Spinal cords from four mice receiving adoptive transfer ofundepleted splenocytes were used in these analyses. The differences between all groups are statistically significant (P , 0.01).

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specific mechanisms responsible for axonal damage in virus-induced demyelination.

ACKNOWLEDGMENTS

Ajai A. Dandekar and Gregory F. Wu contributed equally to thiswork.

We thank Sonya Mehta, Image Analysis Facility, University of Iowa,for helping design methods for quantification of axonal damage.

This research was supported in part by grants from the NationalInstitutes of Health (NS 36592 and NS 40438) and the National Mul-tiple Sclerosis Society (RG2867-A-2). G.F.W. was supported by NRSApredoctoral fellowship MH2066-02.

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FIG. 4. Axonal damage does not precede demyelination. Serial sec-tions (8 mm thick) from the spinal cords of 2.2-V-1-infected RAG12/2mice harvested 4.5 days p.t. were analyzed for demyelination (A),macrophages or microglia (B), viral antigen (C), and nonphosphory-lated NF (D). Examination of the spinal cords of these mice revealedno frank demyelination (A) but abundant viral antigen (C). SMI-32immunoreactivity (D) was detected at low levels and was primarilylocalized to areas of macrophage or microglia infiltration (B). The datashown are representative of analyses of the spinal cords of five mice.Bar, 100 mm.

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